Nucleoside hydrolase and nucleoside phosphorylase detection kits, dipsticks and substrates

ABSTRACT

The invention relates to a method of detecting and/or assaying nucleoside hydrolases or nucleoside phosphorylases using a chromogenic substrate. Preferred chromogenic substrates have formula (I) where X is OH, or H, and Y is the residue of Y—OH where Y—OH is a chromophore or a compound readily converted to a chromophore and the substrates are hydrolyzed by the nucleoside hydrolase to yield ribose or 2-deoxyribose plus Y—OH. Alternatively, those substrates may be phosphorylysed by nucleoside phosphorylase to yield ribose-1-phosphate plus Y—OH. The methods may be used to detect and/or assay parasites in biological samples.

This is a continuation of U.S. application Ser. No. 09/125,808, filedFeb. 22, 1999, now U.S. Pat. No. 6,379,911 B2. which issued on Apr. 30,2002. which is a U.S. national phase filing of PCT Application No.PCT/NZ97/00021, filed Feb. 24, 1997, and claims priority to New ZealandApplication No. 286059, filed Feb. 23, 1996.

The invention relates to an enzyme detection method and to its use indetection of parasites.

BACKGROUND ART

Protozoan parasites have an adverse effect on the health of human andanimal populations in a large number of countries. Problems presented byprotozoan parasites are particularly of concern in tropical areas of theworld where modern diagnostic methods are often not easily accessible.

Nucleoside hydrolases are enzymes which hydrolyse nucleosides betweenthe ribose or deoxyribose and the purine or pyrimidine base groups. Anumber of different types of nucleoside hydrolases are known.N-Ribohydrolases hydrolyse ribonucleosides and N-deoxyribohydrolaseshydrolyse deoxyribonucleosides. Within each of these two general groupsof enzymes are enzymes of differing specificities.

Among the well-characterized N-ribohydrolases, specificity is high forthe ribosyl group but varies for the leaving group purine or pyrimidine.The inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase) fromthe trypanosome Crithidia fasciculata hydrolyzes all of the naturallyoccurring purine and pyrimidine nucleosides with similar catalyticefficiencies. The guanosine-inosine enzyme (GI-nucleoside hydrolase)from the same organism has a strong preference for the eponymoussubstrates and is nearly inert with the pyrimidinenucleosides^(1,2,3,4). AMP Nucleosidase from bacterial sources is highlyspecific for the adenine base, and the 5′-phosphoryl is required forsignificant hydrolytic rates^(5,6). Nucleoside phosphorylases have asimilar mechanism to nucleoside hydrolases and, for example, purinenucleoside phosphorylase is specific for inosine and guanosinesubstrates and activates phosphate or arsenate anions to attack C1 ofthe nucleosides.

It is an object of the invention to provide a method of detecting and/orassaying for the presence of certain enzymes, especially those ofparasites in samples taken from parasitised humans and animals.

SUMMARY OF THE INVENTION

In one aspect the invention provides a method of detecting and/orassaying nucleoside hydrolases using a chromogenic substrate.

Preferably the chromogenic substrates have the formula:

where X is OH or H, and Y is the residue of Y—OH where Y—OH is achromophore or a compound readily converted to a chromophore and thesubstrates are hydrolysed by the nucleoside hydrolase to yield ribose or2-deoxyribose plus Y—OH.

Preferably the chromogenic substrates are of Formula I wherein X and Yare as defined and the substrates are phosphorylysed by the nucleosidephosphorylase to yield ribose-1-phosphate or 2-deoxyribose-1-phosphateplus Y—OH.

Y may be chosen so that Y—OH is a compound absorbing in the visible orUV light, readily measured at wavelengths greater than 300 nm,preferably greater than 340 nm.

Preferably Y—OH is 6-hydroxynicotinamide or2-hydroxypyridine-4-carboxamide.

Y may be chosen so that Y—OH is a chemiluminescent compound eg luminolwhich when released and oxidized by chemical or enzymatic means emitslight.

Alternatively Y—OH may be a compound readily converted to a colouredcompound eg by reaction with a diazonium salt eg α-naphthol.

More preferably Y—OH is a fluorescent compound eg 4-methylumbelliferoneor fluoroscein.

Most preferably Y—OH is a coloured compound eg phenolphthalein,p-nitrophenol, thymolphthalein, 2-nitrophenol,2-hydroxy-5-nitropyridine.

It is preferred that Y—OH can be measured in the presence of thecompound of Formula I by virtue of Y—OH absorbing or fluorescing to agreater extent than the compound of Formula I at certain wavelengths.Generally this will be by virtue of Y—OH ionising and being inequilibrium with quinonoid-like forms.

A particularly preferred substrate is p-nitrophenyl β-D-ribofuranoside.Also particularly preferred is 4-pyridyl β-D-ribofuranoside. Furtherparticularly preferred substrates are 4-methylumbelliferylβ-D-ribofuranoside (4-methylcoumarin-7-yl β-D-ribofuranoside) and2-(5-nitropandyl) β-D-ribofuranoside.

According to another aspect of the invention, there is provided a methodfor detecting and/or assaying for parasites especially protozoa insamples obtained usually from humans or animals using a chromogenicsubstrate, preferably of Formula I.

More preferably the chromogenic substrate is one where Y—OH isfluorescent or coloured.

Another preferred type of substrate is one where Y—OH ischemiluminescent eg luminol.

According to a further aspect of the invention there is provided a kitcontaining materials for detection or assay of hydrolysis of thechromogenic substrate, preferably of Formula I, by enzymes in abiological sample. Preferred kits comprise the chromogenic substrate indry form, together with a buffer. Other components eg a cell-lysingagent may also be included.

According to a further aspect of the invention there is provided adipstick containing a chromogenic substrate, preferably of Formula I,for use in detecting nucleoside hydrolases.

According to a further aspect of the invention there are provided novelcompounds of the invention of formula I. In this aspect of the inventionX and Y are as previously defined except the known compounds α-naphthylβ-D-ribofuranoside, 4-methylcoumarin-7-yl 2-deoxy-β-D-ribofuranoside,p-nitrophenyl β-D-ribofuranoside, p-aminophenyl β-D-ribofuranoside,5-amino-6-chloro-3-pyridazinyl β-D-ribofuranoside, 4-chlorophenylβ-D-ribofuranoside, phenyl β-D-ribofuranoside, 4-methoxyphenylβ-D-ribofuranoside, 4-hydroxyphenyl β-D-ribofuranoside,4-(N,N,N-trimethylammonio)phenyl β-D-ribofuranoside, 4-acetylphenylβ-D-ribofuranoside and the β-D-ribofuranosides of L-DOPA andL-α-methyl-DOPA and the N-acetyl methyl esters of the abovementionedDOPA derivatives are not included in this aspect. The exceptions are notknown as chromogenic substrates or for use in assay of parasites.Generally the chromogenic group is not coloured when present in thecompound of Formula I but is readily detectable when that compound ishydrolysed to give Y—OH.

Preferred compounds are those defined above in which Y is an optionallysubstituted pyridyl group or a nitrophenyl group.

Particularly preferred novel compounds of the invention include3-trifluoroacetamidophenyl β-D-ribofuranoside,3-aminophenyl-β-D-ribofuranoside, 1-tetralone-5-yl β-D-ribofuranoside,3-(4-hydroxyphenyl)-1(3H)isobenzofuranone-3-(phen-4-yl)β-D-ribofuranoside, 2-nitrophenyl β-D-ribofuranoside,4-methylcoumarin-7-yl β-D-ribofuranoside, 3-pyridyl β-D-ribofuranoside,4-pyridyl β-D-ribofuranoside, 2-(5-nitropyridyl) β-D-ribofuranoside,5-quinolyl β-D-ribofuranoside, the β-D-ribofuranoside of luminol,p-nitrophenyl 2-deoxy-β-D-erythro-pentofuranoside,3-carboxamido-6-pyridyl β-D-ribofuranoside, and 4-forymylphenylβ-D-ribofuranoside.

In a further aspect of the invention there is provided a method ofpreparing a chromogenic substrate of the invention.

According to a further aspect the invention may be directed to a methodto detect or assay for the presence of nucleoside phosphorylases using achromogenic substrate.

DETAILED DESCRIPTION OF THE INVENTION

One group of preferred chromogenic substrates have a β-D-ribofuranosylgroup attached through a β-O-ribosidic linkage to a chromogenic group.

In a preferred embodiment the chromogenic substrate is a compound ofFormula I wherein X═OH. The group Y is as defined under Formula I.Preferably the chromophore Y—OH is selected from the group ofphenolphthalein, p-nitrophenol, 4-methylumbelliferone, α-naphthol,thymolphthalein, 2-nitrophenol, 2-hydroxy-5-nitropyridine,6-hydroxynicotinamide, 2hydroxypyridine-4-carboxamide and fluorescein.Other chromophores as will be known in the art may also be used.

In this specification the term “chromogenic group” is used to refer to agroup in a nucleoside hydrolase substrate bound to the sugar moietywhich, when enzymatically removed from the substrate, forms a compoundwhich is readily detectable by its visible or UV absorption or by itsfluorescence. It may be detectable either as released or after pHalteration, or after a subsequent reaction. The term “chromophore” isused for the readily detectable product as released and/or asdetectable. The term “chromogen” as used in chemical names, eg1-O-chromogen derivatives, indicates that the relevant group is a“chromogenic group” as defined above. It is preferred that the“chromophore” is readily detectable by its visible absorption, oralternatively, by its fluorescence, but the definition herein alsoincludes other molecules. The term “chromogenic substrate” is used torefer to a substrate containing a “chromogenic group”.

In general it is preferred that the chromophore Y—OH is coloured or canbe converted to a coloured form simply by altering the pH. Especiallypreferred chromophores include p-nitrophenol, phenolphthalein,thymolphthalein and 2-hydroxy-5-nitropyridine. For applications wherethe enzyme levels are low, a fluorescent chromophore may be preferred,especially 4-methylumbelliferone.

The invention is not limited solely to the detection of nucleosidehydrolases. Nucleoside phosphorylases are not hydrolases as they usephosphate as the nucleophile rather than water. The result is a yield ofribose-1-phosphate rather than ribose. Nucleoside phosphorylases have asimilar mechanism to nucleoside hydrolases and can be detected using themethod of the present invention.

On hydrolysis the chromogenic group is released from the molecule andmay be conveniently measured as the chromophore. Phenolphthaleinreleased by hydrolysis gives a red colour, p-nitrophenol and2-nitrophenol a yellow colour and thymolphthalein a blue colour.4-Methylumbelliferone and fluorescein are intensely fluorescentproducts. α-Naphthol will react with diazonium salts to give highlycoloured dyes when released from the substrate.2-Hydroxy-5-nitropyridine, 6-hydroxynicotinamide and2-hydroxypyridine-4-carboxamide are further compounds which whenreleased from a substrate are readily measurable when the —OH isionised. Intensity of colour or fluorescence increases on making thehydrolysate alkaline to fully ionise the phenolic groups for some of theabove compounds as is well known in the art.

A number of the preferred substrates yield a Y—OH which is anitrophenol, or a hydroxypyridine. Preferred substrates include theβ-D-ribofuranosides of 2-nitrophenol, 4-methyl umbelliferone, 3- and4-hydroxypyridine, 2-hydroxy-5-nitropyridine, luminol,6-hydroxynicotinamide and 4-formylphenol.

A particularly preferred substrate is p-nitrophenyl β-D-ribofuranoside(“nitrophenylriboside”). Another particularly preferred substrate is4-pyridyl β-D-ribofuranoside (“4-pyridylriboside”).

The enzyme assay/detection method may be used in a manner which takesadvantage of the fact that the chromogenic substrates are considerablymore efficiently hydrolysed by some nucleoside hydrolases than others.

In another aspect of the invention chromogenic substrates are hydrolysedby parasite nucleoside hydrolases releasing the chromophore allowing thedetection or assay of the parasite especially in mammalian samples.Among the parasites which may be detected/assayed by this type of methodare Giardia, Trichomonas, Leishmania, Trypanosoma, Crithidia,Herpetomonas, and Leptomonas, especially Trypanosoma. Also especiallypreferred for use in this method are Giardia, Toxoplasma and Neophoramay also be detected/assayed by the method of the invention. As will beapparent to those skilled in the art, the method can be advantageouslyapplied with any parasite containing one or more nucleoside hydrolaseswhich can catalyse hydrolysis of the chromogenic substrate efficientlyenough to be detectable/measurable in a mammalian sample.

Trypanosoma cruzi, Giardia intestinalis, and T. vaginalis areparticularly suitable for assay/detection by the method of theinvention.

In another aspect of the invention, deoxyribonucleoside hydrolases andprotozoan parasites containing deoxyribonucleoside hydrolases can bedetected by analogous methods when the chromogenic substrates usedcontain a 2-deoxy-β-D-ribofuranosyl moiety rather than aβ-D-ribofuranosyl moiety. In preferred embodiments of this aspect thechromogenic substrate is a compound of formula I wherein X═H. Thechromogenic group Y is as defined under formula I. Preferably thechromophore when released from the substrate is selected from one ofphenolphthalein, p-nitrophenol, 4-methylumbelliferone, α-naphthol,thymolphthalein, 2-nitrophenol, 2-hydroxy-5-nitropyridine,6-hydroxynicotinamide, 2-hydroxypyridine-4-carboxamide and fluorescein,especially p-nitrophenol and 4-methylumbelliferone.

Chromogenic substrates may be prepared starting with a ribosyl estersuch as 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose which iscommercially available from Aldrich. This can be coupled to achromophore molecule with a free OH. This may be achieved using a LewisAcid such as BF₃.(OEt)₂ as catalyst to give a 1-O-chromogen derivative.The 1-O-chromogen-triesterified-ribose compound (eg1-O-chromogen-tri-O-benzoyl ribose) can be extracted and purified andthe ester (eg benzoate) groups removed by hydrolysis (eg by stirringovernight in methanol adjusted to about pH 10 with aqueous sodiumhydroxide). If so desired, phosphorylation at the five position of theribofuranose moiety may be accomplished by reaction withN,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine intetrazole-acetonitrile followed by oxidation with m-chloroperoxybenzoicacid. The resulting 5-O-(o-xylylenephosphato)-β-D-ribofuranosidecompound may be converted to the corresponding ribofuranoside5-O-phosphate by hydrogenation e.g. over Pd/C in ethanol followed byneutralisation with aqueous sodium hydroxide.

Reaction scheme 1 includes the above general reaction scheme as MethodA.

Alternative syntheses are also possible, for example, by Michael orKoenigs-Knorr type synthesis involving O-protected ribofuranosyl halides(eg acetates, benzoates) and reaction with phenolate salt or phenol andheavy metal catalyst eg Ag(I). Hg(II) salts or oxide. The halide may bea chloride, bromide or a fluoride (eg see Method B of Reaction Scheme1).

Reagents: (Method A): i, Y—OH, BF₃.OEt₂; ii, NaOH, H₂O, MeOH; (MethodB); iii, TiCl₄, CH₂Cl₂; iv, Y—O⁻Ag⁺, Toluene; v, K₂CO₃, MeOH thenAmberlite IRC-50 (H⁺) resin; vi,N,N-diethyl-1,5-dianhydro-2,4,3-benzodioxaphosphepin-3-amine,1H-tetrazole, then MCPBA; vii, H₂, Pd/C, EtOH, then NaOH.

Y—OH or its silver salt (Y—O⁻Ag⁺) is a chromophore as defined in thespecification.

Reagents: i, MeOH, HCl; ii, 4-MeBzCl, Pyridine; iii, HCl, AcOH; iv,Y—O⁻Na⁺, DMF; v, K₂CO₃, MeOH; vi,N,N-diethyl-1,5-dianhydro-2,4,3-benzodioxa-phosphepin-3-amine,1H-tetrazole, then MCPBA; vii, H₂, Pd/C, EtOH, then NaOH.

Y—O⁻Na⁺ is the sodium salt of Y—OH, a chromophore as defined in thespecification.

Corresponding deoxy-β-ribofuranosyl compounds may be prepared using theabove methods but with the ribofuranosyl starting materials replacedwith the corresponding 2-deoxyribofuranosyl compounds. A preferredmethod is shown in reaction scheme 2.

Generally in the method of the invention for detection/assay ofparasites, the samples to be tested are brought into contact with thesubstrate at an appropriate pH and the reaction is allowed to proceed.The reaction may take place in a spectrophotometer cuvette to which thechromogenic substrate has been added in an appropriate buffer. Thechoice of buffer and pH will be influenced by a number of factorsincluding the pH activity profile of the enzyme and the particularparasites under investigation. The choice of buffer and pH can readilybe determined by those skilled in the art. The pH will generally be inthe range 7-8.5. When a substrate such as nitrophenylriboside orphenolphthalein riboside is being used, use of slightly alkaline pHvalues may be preferred as, when the chromophore is released,p-nitrophenol and phenolphthalein will then be more coloured. Where achromophore is coloured at the pH of the reaction its release from theriboside can be directly monitored in a spectrophotometer for example.Where the reaction is carried out at a pH in which the chromophore isnot coloured, it may be necessary to allow the reaction to proceed forsome time and then adjust the pH to observe the chromophore at a pHwhere it is coloured.

In a preferred method the sample is brought into contact withp-nitrophenylriboside at about pH 8 and the reaction is monitored byappearance of colour of a nitrophenolate ion formation. The colour maybe monitored visually or by measuring absorption at 400 nm.

Where the chromophore is α-napthol, the reaction is allowed to proceedand then after a time the chromophore is reacted with a diazonium saltto produce the coloured compound which is subsequently used to measurethe extent of the reaction.

Where the chromophore is luminol, the luminol released in the enzymereaction is subsequently oxidized by chemical on enzymic means known inthe art and the emitted light from the chemiluminescence obtained ismeasured.

The invention may be practised with many variations so long as thecritical feature of a substrate of a nucleoside hydrolase orphosphorylase, where the base is replaced by a chromogenic group, ispresent. Generally the chromogenic group is not coloured when present onthe substrate but is when released by the action of the enzyme—or elseis otherwise readily detected once liberated from the substrate. Thoseskilled in the art will appreciate that this may be carried out withmany variations.

The reaction need not necessarily be carried out in a test tube orcuvette. The reagents may be included in test strips whereby biologicalsample is added to the test strip and colour is generated if the samplecontains parasites containing one or more enzymes which hydrolyse thechromogenic substrate. For convenience the test strip may form part of adipstick. Such dipsticks may be prepared according to methods well knownin the art.

The invention may be carried out with many sample types, e.g. biologicalsamples including blood or serum samples. The samples may be taken froma mammal (e.g a human, bovine, pig, goat, sheep or horse) but may alsobe taken from other species (eg fish species or from the environment).

For some samples minimal preparation is necessary. For example the assayfor Trypanosoma cruzi, the causative agent of Chagas disease, may becarried out on a blood lysate containing one microliter of infectedblood. The lysate may be prepared by mixing blood with an equal volumeof 1% nonionic detergent. Other sample preparation methods may be used.For example samples may be disrupted by use of sonication.

In another aspect of the invention, advantage can be taken of thedifferent substrate specificity of the different parasite enzymes toassist in determining the nature of a parasite infection. Differences inspecificities are illustrated in the Examples by comparison of kineticdata from IU-nucleoside hydrolase from Crithidia fasciculata andIAG-nucleoside hydrolase from Trypanosoma brucei brucei. The compoundp-nitrophenylriboside is a particularly good substrate for the formerbut not the latter. In contrast, 4-pyridyl riboside and2-(5-nitropyridyl) riboside show lower activity with the former enzymethan does nitrophenylriboside, but substantially greater activity withthe latter than does the nitrophenylriboside.

Kits containing a chromogenic substrate of Formula (I) optionallytogether with a buffer and/or materials for processing the biologicalsample prior to assay/detection (eg a cell-lysing solution) are includedwithin the invention. Preferably the substrate is in a dry form tominimise hydrolysis during storage.

EXAMPLES

The following examples further illustrate practice of the invention.Ratios of solvents are by volume unless otherwise indicated.

Experimental

N.m.r. spectra were recorded on a Bruker AC-300 instrument at 300 MHz(¹H) or 75 MHz (¹³C) and referenced to TMS (¹H nmr, δ 0.00), CD₃OD (¹³Cnmr, δ 49.0), d₆-DMSO (¹³C nmr, δ 40.9) or as otherwise indicated. The¹³C nmr resonance values are followed in brackets by the abbreviationss, d, t or q referring to (C), (CH), (CH₂) or (CH₃) groups respectively,as determined by distortionless enhancement by polarization transfer(DEPT) experiments. For 1H nmr the following abbreviations are used: s(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br(broad).

Example 1 Stage 1—Preparation of p-nitrophenyl2,3,5-tri-O-benzoyl-β-D-ribofuranoside

A solution of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (10 g),p-nitrophenol (5.6 g, 2 eq) and boron trifluoride diethyl etherate (1.2ml, 0.5 eq) in dry dichloromethane (100 ml) was allowed to stand at roomtemperature overnight. Thin-layer chromatography on silica gel [EluantEtOAc:CHCl₃:Hexanes 1:2:4] then indicated essentially completeconversion to a slightly less polar material. The solution was washedwith aq bicarbonate and processed conventionally. Flash chromatographyon silica gel [eluant EtOAc:CHCl₃:Hexanes 1:2:8] then afforded 8.29 g oftitle compound.

Stage 2—Preparation of p-nitrophenyl β-D-ribofuranoside

The above material (8.29 g) was suspended in methanol (100 ml) and thepH was adjusted to about 10 with aq NaOH. After stirring overnight thesolution was homogeneous and thin layer chromatography on silica gel[eluant CHCl₃:EtOAc:MeOH 5:2:1] indicated the reaction was complete. Thesolution was concentrated under reduced pressure, the residue wasdissolved in MeOH/CH₂Cl₂ [about ½ v/v] and filtered through a pad ofsilica gel. The silica was washed if necessary with CHCl₃:EtOAc:MeOH5:2:2 to elute all of the product. Concentration of the eluate affordeda pale yellow solid contaminated with methyl benzoate. Trituration withEtOAc gave the title compound (2.77 g). Recrystallisation from EtOAcgave material with mp 156-158° C.

Example 2 Preparation of α-naphthyl β-D-ribofuranoside Stage 1.Preparation of α-naphthyl 2,3,5-tri-O-benzoyl-β-D-ribofuranoside

A solution 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (500 mg, 0.99mmol). α-naphthol (428 mg. 3.00 mmol) and boron trifluoridediethyletherate (141 mg, 0.99 mmol, 125 μl) in dry dichloromethane (5ml) was stirred at room temperature under argon for 16 hours. Themixture was worked up as for Stage 1 of Example 1 and purified by flashchromatography on silica gel (eluting with dichloromethane:hexanes, 3:2)to afford the title compound contaminated with α-naphthol. The latterwas removed by dissolving the residue in ethyl acetate and washing with10% aqueous sodium hydroxide solution, which after conventionalprocessing, gave 136 mg, 23% of product as a yellow gum.

Stage 2. Preparation of α-naphthyl β-D-ribofuranoside

The product from Stage 1, (130 mg, 0.22 mmol) was dissolved in methanol(3 ml) and the pH adjusted to about 12 by the addition of 10% aqueoussodium hydroxide. After 5 hours, Amberlite IRC•50 (H⁻) resin was addedto neutralize the excess base until pH 5 had been reached, at whichpoint the resin was filtered off. The filtrate was evaporated in vacuoand purified by flash chromatography on silica gel (5% methanol indichloromethane) to afford the title compound as a colourlesscrystalline solid (37 mg, 61%) after trituration with dichloromethane.

¹H nmr (d₆-DMSO) δ ppm: 8.12 (dd, 1H, J=6.8, 2.5 Hz), 7.87 (dd, 1H,J=6.7, 2.3 Hz), 7.54-7.48 (m, 3H), 7.42 (t, 1H, J=7.8 Hz), 7.12 (d, 1H,J=7.5 Hz), 5.67 (s, 1H), 5.40 (d, 1H, J=4.5 Hz), 5.05 (d, 1H, J=6.3 Hz),4.68 (t, 1H, J=5.4 Hz), 4.23 (t, 1H, J=4.1 Hz), 4.19-4.13 (m, 1H),4.00-3.95 (m, 1H), 3.62-3.56 (m, 1H), 3.42-3.37 (m, 1H). ¹³C nmr(d₆-DMSO) δ ppm; 153.4(s), 135.5(s), 128.8 (d), 127.8 (d), 127.5(d),126.8(d), 126.7(s), 123.01(d), 122.3(d), 109.8(d), 107.0(d), 86.1(d),76.2 (d), 72.2(d), 64.2(t).

Example 3 Preparation of 3-trifluoroacetamidophenyl β-D-ribofuranosideStage 1.—Preparation of 3-trifluoroacetamidophenol

A solution of 3-aminophenol (500 mg, 4.58 mmol), trifluoroaceticanhydride (9.62 g, 45.8 mmol. 6.4 ml) and trifluoroacetic acid (10 ml)was stirred at 0° C. for 2 hrs. The excess solvents were evaporated invacuo to a solid residue which was dissolved in ethyl acetate and washedwith saturated aqueous sodium bicarbonate solution. The organic layerwas dried over anhydrous magnesium sulfate, evaporated in vacuo and theresidue recrystallized from toluene to afford the title compound ascolourless needles, 758 mg, 67%, m.p. 133°-135° C.

Stage 2.—Preparation of 3-trifluoroacetamidophenyl2,3,5-tri-O-benzoyl-β-D-ribofuranoside

A solution of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (500 mg,0.99 mmol), 3-trifluoroacetamidophenol (407 mg, 1.98 mmol) from Stage 1and boron trifluoride diethyletherate (1.41 g, 9.93 mmol, 1.25 ml) in amixture of dry dichloromethane (10 ml) and dry tetrahydrofuran (30 ml)was stirred at room temperature under argon for 7 days. Work-up as inStage 1 of Example 1 and purification by flash chromatography on silicagel (2% acetone in toluene as eluant) gave the title compound as acolourless solid (166 mg. 21%).

Stage 3.—Preparation of 3-trifluoroacetamidophenyl β-D-ribofuranoside

The product from Stage 2 (135 mg, 0.21 mmol) was dissolved in methanol(4 ml), 10% aqueous sodium hydroxide was added to raise the pH to about13, and then the mixture was stirred for 89 hours. The mixture wasneutralized with Amberlite IRC-50 (H⁻) resin to pH 5, filtered and thefiltrate evaporated to a solid residue which was further purified byflash chromatography on silica gel (5% methanol in dichloromethanel togive the title compound as a colourless gum (17 mg, 24%).

¹H nmr (CD₃OD) δ ppm; 7.37 (m, 1H), 7.27 (m, 2H), 6.89 (m, 1H), 5.54 (s.1H), 4.22-4.15 (m, 2H), 4.06 (dt, 1H, J=6.4, 3.6 Hz), 3.73 (dd, 1H,J=11.9, 3.6 Hz), 3.55 (dd, 1H, J=11.9, 6.2 Hz). ¹³C nmr (d₆-DMSO) δ ppm;158.3(s), 155.9(s)(J_(F-C)=37 Hz), 138.9(s), 131.2(d), 117.2(s)(J_(F-C)=289 Hz) 115.6(d), 114.9(d), 110.4(d), 106.7(d), 87.2(d),76.0(d), 72.0(d), 64.1(t).

A second product (31 mg) was also obtained which by ¹H nmr consisted ofa 2:1 mixture of the title product together with 3-aminophenylβ-D-ribofuranoside.

Example 4 Preparation of 3-aminophenyl β-D-ribofuranoside

The mixture of 3-trifluoroacetamidophenyl β-D-ribofuranoside and3-aminophenyl β-D-ribofuranoside from Stage 3 of Example 3 (31 mg) wasstirred for 16 hours with 3M aqueous sodium hydroxide, evaporated invacuo then purified by flash chromatography on silica gel (eluting with15% methanol in dichloromethane) giving the title compound as a gum (4mg).

¹H nmr (CD₃OD) δ ppm; 6.96 (t, 1H, J=8.0 Hz), 6.39 (d, 1H, J=2.1 Hz),6.34 (m, 2H), 5.47 (s, 1H), 4.17 (dd, 1H, J=6.7. 4.8 Hz), 4.10 (dd, 1H,J=4.7, 0.9 Hz), 4.02 (dt, 1H, J=6.4, 3.7 Hz), 3.71 (dd, 1H, J=11.9, 3.6Hz), 3.56 (dd, 1H, J=11.8, 6.2 Hz). ¹³C nmr (CD₃OD) δ ppm; 159.2(s),150.2(s), 130.8(d), 110.5(d), 107.1(d), 106.8(d). 104.7(d), 85.6(d),76.6(d), 72.4(d), 64.7(t).

Example 5 Preparation of 1-tetralone-5-yl β-D-ribofuranoside Stage1.—Preparation of 1-tetralone-5-yl2,3,5-tri-O-benzoyl-β-D-ribofuranoside

A solution of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (426 mg,0.84 mmol), 5-hydroxy-1-tetralone (122 mg, 0.75 mmol) and borontrifluoride diethyletherate (120 mg, 0.84 mmol, 106 μl) indichloromethane (4 ml) was stirred at room temperature, under argon for19 hours. Work-up as for Stage 1 in Example 1 then flash chromatographyon silica gel (eluting with 2% acetone in toluene) gave the titlecompound as a colourless oil (308 mg of approx. 51% purity).

Stage 2.—Preparation of 1-tetralone-5-yl β-D-ribofuranoside

The product from Stage 1 (308 mg) was stirred with anhydrous potassiumcarbonate (245 mg, 1.77 mmol) in methanol (5 ml) for 3 hours at roomtemperature. Evaporation of the solvent in vacuo, then flashchromatography on silica gel (eluting with 7% methanol indichloromethane) afforded a colourless solid (77 mg) composed ofapproximately 7 parts title compound and 1 part of the α-anomer of thetitle compound.

¹H nmr (CD₃OD) δ ppm; 7.61 (d, 1H, J=7.7 Hz), 7.35 (dd, 1H, J=8.1. 1.0Hz), 7.25 (t, 1H, J=7.9 Hz), 5.63 (d, 0.13H, J=4.4 Hz, H-1 of α-anomer),5.56 (s, 0.87H, H-1 of β-anomer), 4.28-4.11 (m, 2H), 4.10-4.05 (m, 1H),3.74 (dd, 0.87H, J=11.9, 3.6 Hz), 3.69-3.62 (m, 0.13H), 3.56 (dd, 1H,J=11.9, 6.0 Hz), 3.02 (t, 0.26H, J=6.1 Hz), 2.89 (t, 1.74H, J=6.1 Hz),2.59 (dd, 2H, J=7.0, 6.2 Hz), 2.07 (t, 2H, J=6.4 Hz). ¹³C nmr (CD₃OD) δppm; 201.2(s)_(α,β), 156.7(s)_(α), 156.0(s)_(β), 137.5(s)_(α),136.3(s)_(β), 135.2(s)_(β), 131.0(d)_(α), 128.3(d)_(β), 128.2(d)_(α),122.3 (d)_(α), 121.8(d)_(α), 121.4(d)_(β), 120.9(d)_(β), 107.5(d)_(β),103.5(d)_(α), 88.3(d)_(α), 86.3(d)_(β), 77.1(d)_(β), 73.9 (d)_(α),72.8(d)_(α), 71.6(d)_(α), 64.9(t)_(β), 63.7(t)_(α), 40.2(t)_(α),40.1(t)_(β), 24.6(t)_(α), 24.5(t)_(β), 24.2(t)_(α), 24.1(t)_(β).

Example 6 Preparation of p-aminophenyl β-D-ribofuranoside

The product from Stage 2 of Example 1 (p-nitrophenyl β-D-ribofuranoside31 mg, 0.11 mmol) was suspended in methanol (5 ml) containing 10%palladium on carbon (14 mg) and hydrogenated for 16 hrs. The palladiumcatalyst was filtered off and the solvent evaporated in vacuo to givethe title compound as a pale pink gum (23 mg, 82%).

¹H nmr (CD₃OD) δ ppm, 6.82 (d, 2H, J=8.8 Hz), 6.67 (d, 2H, J=8.8 Hz),5.36 (s, 1H), 4.17 (dd, 1H, J=6.6, 4.8 Hz), 4.10 (dd, 1H, J=4.8, 0.8Hz), 4.02 (dt, 1H, J=6.4, 3.7 Hz), 3.73 (dd, 1H, J=11.8, 3.7 Hz), 3.59(dd, 1H, J=11.8, 6.2 Hz), ¹³C nmr (CD₃OD) δ ppm; 151.4(s), 143.3(s),119.1(d), 117.96(d), 108.2(d), 85.6(d), 76.7(d), 72.6(d), 64.9(t).

Example 7 Preparation of3-(4-hydroxyphenyl)-1(3H)-isobenzofuranone-3-(phen-4-yl)β-D-ribofuranoside (The Mono Phenolphthalein Glycoside of D-ribose)Stage 1.—Preparation of3-(4-hydroxyphenyl)-1(3H)-isobenzofuranone-3-(phen-4-yl)2,3,5-tri-O-benzoyl-β-D-ribofuranoside

A suspension of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (545 mg,1.08 mmol), phenolphthalein (702 mg, 2.21 mmol) and boron trifluoridediethyletherate (153 mg, 1.08 mmol, 136 μl) in dry dichloromethane (6ml) was stirred for 3 hours at room temperature under argon. Morephenolphthalein (611 mg, 1.92 mmol) was added and after a further 3.5hours was worked-up as described in Stage 1 of Example 2 and purified byflash chromatography on silica gel (eluant 7% acetone in toluene). Theproduct (85 mg, 10%) was obtained as a pale yellow oil.

Stage 2.—Preparation of3-(4-hydroxyphenyl)-1(3H)isobenzofuranone-3-(phen-4-yl)β-D-ribofuranoside

The product from Stage 1 (85 mg, 0.11 mmol) and anhydrous potassiumcarbonate were stirred in methanol (2 ml) for 4 hours then worked-up asin Stage 2 of Example 2. The residue was purified by flashchromatography an silica gel (eluting with 7% methanol indichloromethone) to give 32 mg, 64% of a colourless oil as a 4:1 mixtureof title compound and the α-anomer of the title compound.

¹H nmr (CD₃OD) δ ppm; 7.99 (3, 0.21H), 7.89 (d, 0.79H), 7.76 (t, 0.79H),7.63-7.57 (m, 2H), 7.46 (t, 0.21H), 7.22 (d, 2H), 7.08 (d, 2.58H), 7.00(d, 1.42H), 6.74 (d, 2H), 5.63 (d, 0.21H, J=4.4 Hz, H-1 for α-anomer),5.54 (s, 0.79H, H-1 for β-anomer), 4.21-4.02 (m, 3H), 3.73-3.64 (m, 1H),3.56-3.30 (m, 1H). ¹³C nmr (CD₃OD) δ ppm: 171.8(s), 159.0(s), 158.3(s),154.3(s), 135.8(d), 135.7(d), 132.8(s), 130.6(d), 130.4(d), 129.7(d),129.5(d), 129.4(d), 129.3(d), 126.6(d), 126.3(s), 125.6(d), 118.0(d),117.3(d), 116.2(d), 106.7(d), 102.2(d), 93.5(s), 87.6(d), 85.8 (d),76.5(d), 73.3(d), 72.3(d), 71.2(d), 64.6(t), 63.2(t).

Example 8 Preparation of 2,3,5-tri-O-benzoyl-α,β-D-ribofuranosylChloride

To a solution of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (3.02g, 5.98 mmol) in dry dichloromethane (100 ml) was added via cannula adichloromethane solution of titanium tetrachloride (1.13 g, 5.98 mmol,5.98 ml of a 1M solution). The solution was stirred at room temperaturefor 1.5 hours under argon, then washed with water (3×100 ml), dried overanhydrous magnesium sulfate and evaporated in vacuo to give the titlecompounds as a colourless oil (2.9 g, 100%). ¹H nmr indicated it to bean approximately 1:1 mixture of α:β-anomers.

¹H nmr (CDCl₃) δ ppm; 8.06-7.81 (m, 6H), 7.55-7.09 (m, 12H), 6.57 (d,0.5H, J=4.5 Hz, H-1 of α-anomer), 6.20 (s, 0.5H, H-1 of β-anomer), 6.07(dd, 0.5H, J=4.6 Hz), 5.91 (d, 0.5H, J=4.5 Hz), 5.78 (dd, 0.5H, J=7.0,2.9 Hz), 5.48 (dd, 0.5H, J=7.0, 4.6 Hz), 4.96-4.85 (m, 2H), 4.64-4.55(m, 1H).

Example 9 Preparation of 2-nitrophenyl β-D-ribofuranoside Stage1.—Preparation of 2-nitrophenol, Silver Salt

Silver(II) nitrate (849 mg, 5.00 mmol) was dissolved in water (3 ml) andadded to a solution of 2-nitrophenol (696 mg, 5.00 mmol) in 10 ml ofaqueous sodium hydroxide (200 mg, 5.00 mmol) solution. The precipitatewas filtered, washed with diethyl ether and dried in vacuo overphosphorus pentoxide to afford the title compound as an orange powder(1.10 g, 89%).

Stage 2.—Preparation of 2-nitrophenyl2,3,5-tri-O-benzoyl-β-D-ribofuranoside

2,3,5-Tri-O-benzoyl-α,β-D-ribofuranosyl chloride (320 mg, 0.66 mmol, 1eq), prepared in Example 8, was azeotropically dried with toluene,redissolved in dry toluene (20 ml) and added to an azeotropically dried(with toluene) suspension of the silver salt (332 mg, 1.35 mmol 2 eq)prepared in Stage 1, in toluene (20 ml) and refluxed under argon for 1.5hours. The mixture was filtered and the solvent evaporated in vacuo to ayellow oil which was further purified by flash chromatography on silicagel (eluting with dichloromethane:hexanes, 5:1 to give the titlecompound as a gum (226 mg).

Stage 3.—Preparation of 2-nitrophenyl β-D-ribofuranoside

The product from Stage 2 was dissolved in methanol (6 ml), the pHadjusted to 11 by the addition of 10% aqueous sodium hydroxide solutionand stirred for 16 hrs. Work-up as in Stage 2 of Example 2 followed bypurification by flash chromatography on silica gel (eluting with 10%methanol in dichloromethane) afforded the title compound as pale yellowcrystals (62 mg, 61%).

¹H nmr (CD₃OD) δ ppm: 7.78 (dd, 1H, J=8.1, 1.6 Hz), 7.56 (dt, 1H, J=7.9,1.6 Hz), 7.42 (dd, 1H, J=8.5, 0.9 Hz), 7.12 (dt, 1H, J=8.3, 1.1 Hz),5.68 (s, 1H), 4.22 (m, 2H), 4.10 (dt, 1H, J=6.6, 3.5 Hz), 3.74 (dd, 1H,J=11.9, 3.5 Hz), 3.55 (dd, 1H, J=11.9, 6.6 Hz). ¹³C nmr (CD₃OD) δ ppm,150.6(s), 142.2(s), 134.9(d), 125.9(d), 122.8(d), 118.7(d), 107.2(d),86.2(d), 76.3(d), 72.2(d), 64.6(t).

Example 10 Preparation of 4-methylcoumarin-7-yl β-D-ribofuranoside(4-methylumbelliferyl β-D-riboside) Stage 1.—Preparation of7-hydroxy-4-methylcoumarin, Silver Salt

This compound was prepared from 471 mg of 7-hydroxy-4-methylcoumarin bythe method described in Stage 1 of Example 9 to give the title compoundas a brown powder (652 mg, 86%).

Stage 2.—Preparation of 4-methylcoumarin-7-yl2,3,5-tri-O-benzoyl-β-D-ribofuranoside

The title compound was prepared by reaction of the product of Stage 1with the chloride from Example 8 by the method described in Stage 2 ofExample 9 but with a reflux time of 30 hrs. The product was purified byflash chromatography on silica gel (eluting with 1% acetone indichloromethane) to afford 102 mg, 25% of a yellow oil.

Stage 3.—Preparation of 4-methylcoumarin-7-yl β-D-ribofuranoside

The product from Stage 2. (102 mg, 0.16 mmol) was dissolved in methanol(7 ml) and treated with sodium methoxide (35 mg, 0.66 mmol, i.e. 140 mgof a 25% solution in methanol). After 4 hrs the reaction was worked-upas described in Stage 2 of Example 2. Purification by flashchromatography (eluting with 10% methanol in dichloromethane) gave thetitle compound as a colourless solid (24 mg, 47%).

¹H nmr, (d₆-DMSO) δ ppm: 7.70 (d, 1H), 6.98 (m, 2H), 6.24 (s, 1H), 5.60(s, 1H), 5.39 (d, 1H), 5.05 (d, 1H), 4.66 (t, 1H), 4.05 (m, 2H), 3.93(m, 1H), 3.53 (m, 1H), 3.40 (m, 1H), 2.40 (s, 3H). ¹³C nmr (d₆-DMSO) δppm; 161.1(s), 160.5(s), 159.6(s), 154.5(s), 127.7(d), 115.2(s),114.6(d), 112.8(d), 106.4(d), 104.5(d), 86.1(d), 75.8(d), 71.7(d),63.8(t), 19.3(q).

Example 11 Preparation of 3-pyridyl β-D-ribofuranoside Stage1.—Preparation of 3-hydroxypyridine, Silver Salt

This compound was prepared from 500 mg of 3-hydroxypyridine by themethod described in Stage 1 of Example 9 to give the title compound as acream-coloured product (996 mg, 94%).

Stage 2.—Preparation of 3-pyridyl 2,3,5-tri-O-benzoyl-β-D-ribofuranoside

The title compound was prepared by reacting the product from Stage 1with the chloride from Example 8 by the method described in Stage 2 ofExample 9 but with a reflux time of 24 hours. The product was purifiedby flash chromatography on silica gel (eluting with 4% acetone indichloromethane) to afford 144 mg, 44% of a yellow oil.

Stage 3.—Preparation of 3-pyridyl β-D-ribofuranoside

The product from Stage 2 (120 mg, 0.22 mmol) was dissolved in methanol(10 ml), 10% aqueous sodium hydroxide solution added to bring the pH to11, then stirred for 21 hours. Work-up as described in Stage 2 ofExample 2 followed by flash chromatography on silica gel (eluting with10% methanol in dichloromethane) gave the product as a yellow gum (29mg, 57%).

¹H nmr (D₂O) δ ppm; 8.43 (m, 2H), 7.75-7.72 (m, 1H), 7.66-7.58 (m, 1H),5.90 (s, 1H), 4.59-4.53 (m, 2H), 4.35-4.29 (m, 1H), 3.98 (dd, 1H,J=12.4, 3.3 Hz), 3.78 (dd, 1H, J=12.4, 6.2 Hz) (referenced to HOD at4.94 ppm). ¹³C nmr (D₂O) δ ppm; 155.6(s), 145.4(d), 141.1(d), 134.2(s),131.7(s), 131.2(s), 128.2(d), 127.9(d), 107.9(d), 86.7(d), 77.5(d),73.4(d), 65.3(t);

Example 12 Preparation of 4-pyridyl β-D-ribofuranoside Stage1.—Preparation of 4-hydroxypyridine, Silver Salt

This compound was prepared from 243 mg of 4-hydroxypyridine by themethod described in Stage 1 of Example 9 to give the title compound as abrown powder (428 mg. 83%).

Stage 2.—Preparation of 4-pyridyl 2,3,5-tri-O-benzoyl-β-D-ribofuranoside

The title compound was prepared by reacting the product from Stage 1with the chloride from Example 8 by the method described in Stage 2 ofExample 9 but with a reflex time of 3 days. The product was purified byflash chromatography on silica gel (eluting with 4% acetone indichloromethane) to afford the product as a colourless solid (112 mg,38%).

Stage 3.—Preparation of 4-pyridyl β-D-ribofuranoside

The product from Stage 2 (112 mg, 0.21 mmol) was dissolved in methanol(5 ml), the pH was adjusted to 11 with 10% aqueous sodium hydroxidesolution and the mixture was then stirred for 24 hours. Work-up as inStage 2 of Example 2 then purification by flash chromatography on silicagel (eluting with 15% methanol in dichloromethane) gave the titlecompound as a colourless solid (15 mg, 32%).

¹H nmr (CD₃OD) δ ppm; 8.25 (br.d, 2H), 6,96 (d, 2H, J=6.3 Hz), 5.57 (s,1H), 4.14-4.09 (m, 2H), 3.99 (dt, 1H, J=6.0, 3.5 Hz), 3.61 (dd, 1H,J=12.0, 3.4 Hz), 3.40 (dd, 1H, J=12.0, 6.0 Hz). ¹³C nmr (CD₃OD) δ ppm,165.1(s), 151.2(d), 113.3(d), 106.0(d), 86.2(d), 76.3(d), 72.0(d),64.1(t).

Example 13 Preparation of 2-(5-nitropyridyl) β-D-ribofuranoside Stage1.—Preparation of 2-hydroxy-5-nitropyridine, Silver Salt

This compound was prepared from 295 mg of 2-hydroxy-5-nitropyridine bythe method described in Stage 1 of Example 9 to give the title compoundas a green solid (397 mg, 76%).

Stage 2.—Preparation of 2-(5-nitropyridyl)2,3,5-tri-O-benzoyl-β-D-ribofuranoside

The title compound was prepared by reacting the compound from Stage 1with the chloride from Example 8 by the method described in Stage 2 ofExample 9 but with a reflux time of 4 hours. The product was purified byflash chromatography on silica gel (eluting withdichloromethane:hexanes, 4:1) to afford the title compound as colourlesscrystals (220 mg, 68%).

Stage 3.—Preparation of 2-(5-nitropyridyl) β-D-ribofuranoside

The product from Stage 2 (220 mg, 0.35 mmol) was dissolved in methanol(7 ml), 10% aqueous sodium hydroxide solution was added to adjust the pHto 11 and the mixture was stirred 69 hours. Work-up as in Stage 2 ofExample 2 then flash chromatography on silica gel (eluting with 5%methanol in dichloromethane) gave the title compound as a pale yellowsolid (10 mg, 10%).

¹H nmr (CD₃OD) δ ppm; 9.05 (d, 1H, J=2.8 Hz), 8.48 (dd, 1H, J=9.1, 2.8Hz), 6.96 (d, 1H, J=9.2 Hz), 6.39 (s, 1H), 4.27 (dd, 1H, J=7.2, 4.7 Hz),4.19 (d, 1H, J=4.7 Hz), 4.07 (ddd, 1H, J=7.2, 5.7, 3.3 Hz), 3.76 (dd,1H, J=12.1, 3.3 Hz), 3.58 (dd, 1H, J=12.1. 5.7 Hz). ¹³C nmr (CD₃OD) δppm; 166.8(s), 145.3(d), 141.5(s), 135.7(d), 112.7(d), 104.6(d),85.9(d), 76.0(d), 71.7(d), 63.7(t).

Example 14 Preparation of 5-quinolyl β-D-ribofuranoside Stage1.—Preparation of 5-hydroxyquinoline, Silver Salt

This compound was prepared from 236 mg of 5-hydroxyquinoline by themethod described in Stage 1 of Example 9 to give the title compound as ablack powder (373 mg, 91%).

Stage 2.—Preparation of 5-quinolyl2,3,5-tri-O-benzoyl-β-D-ribofuranoside

The title compound was prepared by reaction of the product of Stage 1with the chloride from Example 8 by the method described in Stage 2 ofExample 9 but with a reflux time of 26.5 hours. The filtrate from thereaction was washed with 10% aqueous sodium hydroxide solution thenpurified by flash chromatography on silica gel (eluting with ethylacetate:hexanes, 4:6) to afford the crude title compound as a yellow oil(48 mg).

Stage 3.—Preparation of 5-quinolyl β-D-ribofuranoside

The product from Stage 2 (48 mg, 0.08 mmol) was dissolved in methanol (1ml) and stirred 3 hours with anhydrous potassium carbonate (18 mg).Work-up as in Stage 2 of Example 2 followed by flash chromatography onsilica gel (eluting with 10% methanol in dichloromethane) gave the titlecompound as a pale yellow solid (11 mg. 50%).

¹H nmr (CD₃OD+D₂O) δ ppm; 8.83 (dd, 1H, J=4.3, 1.6 Hz), 8.63 (dd, 1H,J=8.5, 1.6 Hz), 7.70-7.68 (m, 2H), 7.53 (dd, 1H, J=8.5, 4.4 Hz), 7.27(dd, 1H, J=5.8, 3.0 Hz), 5.81 (s. 1H), 4.43-4.38 (m, 2H), 4.17 (dt, 1H,J=5.9, 3.7 Hz), 3.78 (dd, 1H, J=12.0. 3.6 Hz), 3.59 (dd, 1H, J=12.0, 3.7Hz). ¹³C nmr (CD₃OD+D₂O) δ ppm; 153.4(s), 151.5(d), 149.4(s), 133.0(d),131.3(d), 122.5(s), 122.4(d), 122.0(d), 110.3(d), 107.0(d), 85.8(d),76.6(d), 72.4 (d), 64.4(t).

Example 15 Preparation of β-D-ribofuranoside of Luminol Stage1.—Preparation of 3-aminophthalhydrazide, Silver Salt

This compound was prepared from 423 mg of 3-aminophthalhydrazide(luminol) by the method described in Stage 1 of Example 9 to give thetitle compound as a light green powder (575 mg).

Stage 2.—Preparation of 2,3,5-tri-O-benzoyl β-D-ribofuranoside ofLuminol

The title compound was prepared by reaction of the product of Stage 1with the chloride from Example 8 by the method described in Stage 2 ofExample 9 but with a reflux time of 2 hrs. Work-up as in Stage 2 ofExample 9 followed by flash chromatography on silica gel (eluting withethyl acetate:hexanes, 7:13) gave the title compound as a yellow gum(155mg. 17%).

Stage 3.—Preparation of the β-D-ribofuranoside of Luminol

The product from Stage 2 (129 mg, 0.21 mmol) and anhydrous potassiumcarbonate were stirred in methanol (2.5 ml) for 3.5 hours then worked-upas in Stage 2 of Example 2. The residue was purified by flashchromatography on silica gel (eluting with 15% methanol indichloromethane) to give the title compound as a pale yellow oil (35 mg,55%).

¹H nmr (CD₃OD) δ ppm; 7.47 (t, 1H, J=8.0 Hz), 7.01 (d, 1H, J=7.6 Hz),6.92 (d, 1H, J=8.1 Hz), 6.20 (s, 1H), 4.34 (dd, 1H, J=7.1, 4.7 Hz), 4.26(d, 1H, J=4.6 Hz), 4.13 (dt, 1H, J=6.5, 3.3 Hz), 3.81 (dd, 1H, J=12.0,3.2 Hz), 3.62 (dd, 1H, J=12.0, 6.0 Hz). ¹³C nmr (CD₃OD) δ ppm; 164.0(s),152.2(s), 151.3(s), 135.6(d), 127.3(s), 118.2(d), 111.8(s), 110.6(d),104.2(d), 85.7(d), 76.0(d), 72.0(d), 64.2(t).

Example 16 Preparation of2-deoxy-3,5-di-O-toluoyl-α-D-erythro-pentofuranosyl Chloride

A solution of 2-deoxy-D-ribose (5.00 g 37.3 mmol) in dry methanol (95ml) was treated with dry methanol (95 ml) to which acetyl chloride (0.85ml) had been added. The mixture was stirred for 10 minutes, pyridine (15ml) was then added and the mixture was evaporated in vacuo to a yellowoil. The residue was dissolved in dry pyridine (30 ml), cooled to 0° C.and p-toluoyl chloride (12.69 g, 82.1 mmol, 10.8 ml) was added. Themixture was allowed to warm to room temperature and stirred overnight.The reaction mixture was partitioned between ethyl acetate (500 ml) and10% aqueous sulfuric acid (300 ml), and the organic layer was separatedand washed with saturated aqueous sodium bicarbonate solution, driedover anhydrous magnesium sulfate and evaporated in vacuo to a yellowoil. The oil was dissolved in glacial acetic acid (100 ml) to which 5.96g of hydrogen chloride had been added. After 30 minutes the resultingcrystalline solid was filtered, washed with cold diethyl ether and driedto yield the title compound (6.9 g). A portion of this product (2.02 g)was recrystallized from hot toluene to afford the title compound ascolourless crystals (1.90 g).

¹H nmr (CDCl₃) δ ppm; 7.99 (d, 2H, J=8.2 Hz), 7.90 (d, 2H, J=8.2 Hz),7.25 (m, 4H), 6.47 (d, 1H, J=5.0 Hz), 5.56 (dd, 1H, J=7.2, 2.8 Hz), 4.86(m, 1 H), 4.68 (dd, 1H, J=12.1, 3.2 Hz), 4.59 (dd, 1H, J=12.1, 4.3 Hz),2.92-2.82 (m,1H), 2.74 (d, 1H, J=14.8 Hz), 2.42 (s, 3H), 2.41 (s, 3H).¹³C nmr (CDCl₃) δ ppm: 166.4(s), 166.1(s), 144.3(s), 144.0(s), 129.9(d),129.7(d), 129.23(d), 129.20(d), 129.1(d), 126.8(s), 126.7(s), 95.3(d),84.7(d), 73.6(d), 63.5(t), 44.5(t), 21.71(q), 21.67(q).

Example 17 Preparation of p-nitrophenyl2-deoxy-β-D-erythro-pentofuranoside Stage 1.—Preparation ofp-nitrophenyl 2-deoxy-3,5-di-O-toluoyl-β-D-erythro-pentofuranoside

p-Nitrophenol (178 mg, 1.28 mmol) and sodium hydride (64 mg of a 60%dispersion in mineral oil, 1.6 mmol) were stirred together in drydimethylformamide (4 ml) at room temperature for 30 minutes then thechloride (416 mg, 1.07 mmol) from Example 16was added. The mixture wasstirred for a further 3 hours, then water (100 ml) was added and themixture was evaporated in vacuo. The residue was purified by flashchromatography on silica gel (eluting with 20% ethyl acetate in hexanes)to give a pale yellow oil which crystallized from a mixture ofchloroform and hexanes to give the title compound as a colourless solid(190 mg. 36%).

Stage 2.—Preparation of p-nitrophenyl2-deoxy-β-D-erythro-pentofuranoside

The product from Stage 1 (147 mg, 0.30 mmol) and anhydrous potassiumcarbonate (105 mg) were stirred together in methanol (4 ml) for 5 hours.The mixture was evaporated in vacuo and the residue purified by flashchromatography on silica gel (eluting with 7% methanol indichloromethane). The title compound was obtained as a yellow solid (46mg, 61%).

¹H nmr (CD₃OD) δ ppm; 8.18 (d, 2H, J=9.3 Hz), 7.16 (d, 2H, J=9.3 Hz),6.01 (dd, 1H, J=5.3, 2.2 Hz), 4.44 (m, 1H), 3.99 (m, 1H), 3.60 (dd, 1H,J=11.6, 5.3 Hz), 3.49 (dd, 1H, J=11.7, 6.3 Hz), 2.50 (ddd, 1H, J=13.7,6.7, 2.2 Hz), 2.30 (dt, 1H, J=13.8, 5.7 Hz). ¹³C nmr (CD₃OD) δ ppm;163.5(s), 143.2(s), 126.4(d), 117.4(d), 103.6(d), 89.2(d), 72.2(d),64.2(t), 42.2(t).

Example 18 Preparation of4-methylcoumarin-7-yl2-deoxy-β-D-erythro-pentofuranoside Stage1.—Preparation of 4-methylcoumarin-7-yl2-deoxy-3,5-di-O-toluoyl-β-D-erythro-pentofuranoside

7-Hydroxy-4-methylcoumarin (226 mg, 1.28 mmol) and sodium hydride (64 mgof a 60% dispersion in mineral oil, 1.6 mmol) were stirred at roomtemperature for 30 minutes in dry dimethylformamide (4 ml). Then thechloride (423 mg, 1.09 mmol) from Example 16 was added and after 24hours the mixture was processed as in Stage 1 of Example 17 and theproduct isolated by flash chromatography on silica gel (eluting with 2%acetone in dichloromethane) to afford an oil which crystallized from hotmethanol as colourless crystals (305 mg, 53%). ¹H nmr indicated them tobe a 91:9 mixture of the title compound and its α-anomer.

Stage 2.—Preparation of 4-methylcoumarin-7-yl2-deoxy-β-D-erythro-pentofuranoside

The compound from Stage 1 (280 mg, 0.53 mmol) was stirred in methanol (5ml) with anhydrous potassium carbonate (213 mg, 1.53 mmol) at roomtemperature for 63 hours. The reaciton mixture was then evaporated invacuo and the product was isolated by flash chromatography on silica gel(eluting with 7% methanol in dichloromethane). The residue obtained wasrecrystallized from methanol-chloroform to afford the title compound ascolourless needles (64 mg, 41%).

¹H nmr (d₆-DMSO) δ ppm; 7.68 (d, 1H), 6.99 (m, 2H), 6.23 (d, 1H), 6.02(dd, 1H), 5.20 (d, 1H), 4.70 (t, 1H), 4.28 (m, 1H), 3.85 (m, 1H), 3.42(m, 1H), 3.28 (m, 1H), 2.40 (d, 3H), 2.37 (m, 1H), 2.20 (m, 1H). ¹³C nmr(d₆-DMSO) δ ppm; 161.3(s), 160.9(s), 155.5(s), 154.5(s), 127.6(d),115.0(s), 114.7(d), 112.7(d), 104.6(d), 103.4(d), 89.4(d), 71.6(d),63.8(t), 42.2(t), 19.3(q),

Example 19 Preparation of 3-carboxamido-6-pyridyl β-D-ribofuranosideStage 1.—Preparation of 6-hydroxynicotinamide

A solution of 6-hydroxynicotinic acid (1.00 g, 7.19 mmol) andconcentrated sulfuric acid (0.47 ml) in methanol (80 ml) was refluxedfor 10 hours then poured into water and sodium bicarbonate (1.45 g) wasadded. The solvents were evaporated in vacuo and the residue waspurified by flash chromatography on silica gel (eluting with 10%-20%methanol in dichloromethane) to give 6-hydroxynicotinic acid, methylester as a colourless solid (996 mg, 90%). The product was dissolved inconcentrated aqueous ammonia solution and heated at 60° C. for 10 hours.Evaporation of the solvent in vacuo left a solid which wasrecrystallized from water to give 371 mg, 61% of the title compound ascolourless needles.

Stage 2.—Preparation of 6-hydroxynicotinamide, Silver Salt

This compound was prepared from 247 mg of 6-hydroxynicotinamide by themethod described in Stage 1 of Example 9 to give the title compound as apeach-coloured powder (430 mg 98%).

Stage 3.—Preparation of 3-carboxamido-6-pyridyl2,3,5-tri-O-benzoyl-β-D-ribofuranoside

The title compound was prepared by reaction of the product of Stage 2with the chloride of Example 8 by the method described in Stage 2 ofExample 9 but with a reflux time of 3 days then 3.5 days at roomtemperature. The product was purified by flash chromatography on silicagel (eluting with 13% acetone in dichloromethane) to afford 82 mg, 24%of the title compound as a yellow oil.

Stage 4.—Preparation of 3-carboxamido-6-pyridyl β-D-ribofuranoside

The product from Stage 3 (82 mg, 0.14 mmol) was stirred with anhydrouspotassium carbonate (20 mg, 0.15 mmol) in methanol (10 ml) at roomtemperature for 19 hours. The mixture was concentrated in vacuo thenpurified by flash chromatography on silica gel (eluting with 20%methanol in dichloromethane) affording the title compound as a paleorange oil (22 mg, 58%).

¹H nmr (CD₃OD) δ ppm; 8.67 (d, 1H, J=2.1 Hz), 8.17 (dd, 1H, J=8.7, 2.5Hz), 6.88 (dd, 1H, J=8.9, 0.4 Hz), 6.31 (s, 1H), 4.26 (dd, 1H, J=7.1.4.7 Hz), 4.19 (d, 1H, J=4.8 Hz), 4.07 (dt, 1H, J=6.5, 3.4 Hz), 3.76 (dd,1H, J=12.0, 3.4 Hz), 3.58 (dd, 1H, J=12.0, 6.0 Hz). ¹³C nmr (CD₃OD) δppm; 170.0(s), 165.6(s), 148.6(d), 140.1(d), 125.3(s), 112.0(d),104.3(d), 85.8(d), 76.2(d), 72.1(d), 64.2(t).

Example 20 Preparation of 4-formylphenyl β-D-ribofuranoside

A suspension of 1-O-acetyl-2,3,5-tri-O-benzoyl β-D-ribofuranose (1 g,2.0 mmol), p-hydroxybenzaldehyde (0.5 g, 4.1 mmol), and borontrifluoride diethyletherate (0.12 ml, 1 mmol) in 20 ml of drydichloromethane was stirred overnight at room temperature. The solventwas removed in vacuo and the residue extracted with ethyl acetate andbrine. The organic layer was dried with sodium sulfate, filtered and thesolvents were removed in vacuo. The residue was not purified further,but suspended in methanol and the pH adjusted to 11 with 1.0 N aqueousNaOH. This was stirred and with occasional readjustment of the pH untilt.l.c. on silica gel (eluant 1:1 ethyl acetate/hexanes) indicated onlymaterial at the solvent front or baseline. Silica gel was added directlyto the reaction material and the solvent removed in vacuo. The powderwas added to a column of silica gel and purified by flash chromatographywith 100% ethyl acetate as eluant. The title compound was obtained as acolourless syrup.

¹H nmr 200 MHz (d₄-MeOH) δ ppm; 9.90 (1H, s), 7.91 (2H, d), 7.22 (2H,d), 5.73 (1H s), 4.3-4.1 (3H, m), 3.77 (1H, dd), 3.57 (1H, dd).

Example 21 Comparison of Kinetic Parameters for N-ribohydrolases andPurine Nucleoside Phosphorylase with Nucleosides and Nitrophenylriboside

In this Example kinetic parameters for different enzymes were comparedusing nitrophenylriboside or a purine substrate. The results are shownin Table 1. Nitrophenylriboside was a good substrate for theinosine-uridine nucleoside hydrolase of the protozoan parasite Crithidiafasciculata but not for the guanosine-inosine nucleoside hydrolase fromthe same species. A nucleoside hydrolase from Trypanosoma brucei brucei(IAG-nucleoside hydrolase) was relatively poor for hydrolysingnitrophenylriboside. The E. coli and bovine spleen enzymes tested wereineffective in hydrolysing the substrate. The striking ability of one ofthe protozoan parasite enzymes to hydrolyse the substrate provides thebasis for an assay for the protozoan parasites (see Example 22).

Example 22 Assay for Parasites Using Hydrolysis of Nitrophenylriboside

The utility of nitrophenylriboside as a substrate for detectingprotozoan infections using the presence of nucleoside hydrolases wastested using blood from mice infected with Trypanosoma cruzi, thecausitive agent of Chagas disease (Table 2). The substrate readilydetects the presence of the nucleoside hydrolase in a blood lysateprepared from one microliter of infected blood. The values obtained fromthe assay from the infected mice were strikingly elevated above those ofthe uninfected controls.

Example 23 Comparison of Substrates with Different N-ribohydrolaseEnzymes

The enzymes used were highly purified samples from Crithidia fasciculata(IU-nucleoside hydrolase) and Trypanosoma brucei brucei (IAG-nucleosidehydrolase), available as stock solutions of approximately 20 mgprotein/mL.

Each reaction was conducted in a quartz cuvette with a 1 cm path length,placed in the observation chamber of a UV spectrophotometer, and in atotal reaction volume of 600 μL. The temperature was adjusted by use ofa circulating water bath, set so that the cuvette temperatureequilibrated to 30° C. in about 5 minutes.

TABLE 1 Kinetic Parameters for N-Ribohydrolases and Purine NucleosidePhosphorylase with Nucleosides and Nitrophenyl Riboside^(a) NitrophenolRiboside^(R) Purine (or 5-Phosphate)^(P) substrate^(b) enzyme k_(cat)K_(m) k_(cat)/K_(m) substrate k_(cat) K_(m) k_(cat)/K_(m) k_(cat)/K_(m)ratio (s⁻¹) (μM) (M⁻¹s⁻¹) (s⁻¹) (μM) (M⁻¹s⁻¹) IU-nucleoside 239 ± 58 ±4.1 × 10⁶ inosine 28 380  7.6 × 10⁴ 54 hydrolase^(R,c) 32 23IAG-nucleoside 0.82 ± 560 ± 1.5 × 10³ inosine 34 18 1.9 × 10⁶ 8 × 10⁻⁴hydrolase^(R,d) 0.03 50 GI-nucleoside 0.07 ± 468 ± 1.4 × 10² guanosine231  77 3.2 × 10⁶ 4 × 10⁻⁵ hydrolase^(R,c) 0.01 130 AMP 0.0004 ± 6250 ±6.2 × 10⁻² AMP 27 150  1.8 × 10⁵ 3 × 10⁻⁷ nucleosidase^(P,e) 0.0001 1700Purine nucleoside 0.00020 ± 224 ± 8.9 × 10⁻¹ inosine 12 19 6.3 × 10⁵ 1 ×10⁻⁶ phosphorylase^(R,f) 0.00004 76 ^(a)The enzymes were highly purifiedsamples from Crithidia fasciculata ^(c,5,6), Trypanosoma, brucei brucei^(d), E coli ^(e,8,9) and bovine spleen^(f,10). Assays were at pH 8.0 in50 mM HEPES, 30° C. For the allosteric AMP nucelosidase, MgATP waspresent at 100 μM. Purine nucleoside phosphorylase was assayed forphosphorolysis by including 3 mM phosphate in assay mixtures. Thesuperscripts^(R) and ^(P) refer to nitrophenylriboside andnitrophenylriboside 5-phosphate, respectively, as substrates.^(b)Substrate specificity, for purine substrates, the kinetic constantsand their standard errors are available in references 5, 6, 11, 9, 10and 12. The k_(cat)/K_(m) ratio compares the k_(cat)/K_(m) fornitrophenylriboside to that for the indicated purine substrate

The β-D-ribofuranoside substrates were dissolved in aqueous buffer: 50mM CHES for reactions at pH 10, 50 mM HEPES for reactions at pH 8. Forreactions catalyzed by the IU-nucleoside hydrolase and for those of theIAG-nucleoside hydrolase with the substrates of Examples 1 and 12, fiveinitial substrate concentrations in the range 0.3-3 times the K_(m)values were used. For the remaining reactions catalysed by IAGhydrolase, a single substrate concentration of between 100 and 300 μMwas used.

The absorbance of each reaction mixture was monitored at or near theabsorption maximum for the phenolic aglycon (as shown in Table 3).Monitoring began 5-10 minutes before the enzyme was added to determineif the substrate was stable under the reaction conditions. Only with5-nitro-2-pyridyl β-D-ribofuranoside (Example 13) at pH 10 washydrolysis observed in the absence of enzyme, and this background ratewas subtracted in the subsequent calculation of this compound'senzyme-catalyzed reaction rate at this pH.

Reactions were initiated by addition of 2 to 20 μL aliquots of enzymesolution taken either directly from the stock solution or after dilutionin the buffer of the assay mixture, such that absorbance changes of0.005 to 0.080 per minute were observed for compounds active assubstrates. Data were fitted to the Michaelis-Menton equation usingkaleidagraph, and the resulting kinetic parameters along with otherrelevant data are summarized in Table 3.

It can be seen that while the β-D-ribofuranosides of Examples 1, 9, 10,12, 13 and 19 are all good substrates for the IU-nucleoside hydrolase,only those of Examples 12, 13 and 19 are good substrates for theLAG-nucleoside hydrolase. These substrates can therefore be used todistinguish nucleoside hydrolase isozymes, and consequently provideorganism-specific detection.

TABLE 2 Rate of Hydrolysis of Nitrophenyl Riboside with Control orInfected Mouse Blood rate^(b) Sample^(a) (pmol/min/mg) uninfectedcontrol  0.7 ± 0.8 (11) infected, T cruzi var Brazil 4.6 ± 0.5 (3)infected, T cruzi var Tulahuen 3.9 ± 0.5 (4) ^(a)Blood samples wereobtained from uninfected or infected mice, diluted with an equal volumeof 0.5% Triton-X-100 and stored overnight at 5° C. prior to assay.^(b)Reaction mixtures containing 50 mM HEPES pH 8.0 at 30° C. were mixedwith 2 μl of diluted blood sample and the background rates recorded.Reactions were initiated by the addition of nitrophenyl riboside to 0.24mM. The rate of p-nitrophenolate ion formation was monitored at 400 nm,in reaction mixture of 1.0 ml. The numbers in parenthesis are the numberof independent rate determinations.

TABLE 3 KINETIC PARAMETERS FOR TWO N-RIBOHYDROLASES WITH VARIOUS β-D-RIBOFURANOSIDES ΔOD/ ΔOD/ IAG NH¹ mM mM ε for rate (min⁻¹, λmax λmaxY-OH IU NH¹ at 200 μM (nm)³ (nm)³ λmax⁴ At pH 10 substrate At pH 10 pKafor pH pH pH kcat Km Ki pH pH kcat Km Example No. Y-OH 8.0 10.0 10.0(sec⁻¹)² (μM) (μM) 8.0 10.0 (min⁻¹) (μM)  1 7.14 17.0 290 110 0.05 0.19529 400  2 9.34 5.04 .009 7.3 <0.05 332 at 250 μM  3 ? ? yes but 27.7 ?very slow  4 9.87 .74 0.7 72 <0.02 295 at 1240 μM  5 0.64 1.83 1.86 0.5<0.01 <0.01 332 361 360 at 200 μM  6 2.02 <0.01 298  7 29.2 19.2 10 at<0.01 <0.01 553 553 200 μM  9 7.23 4.5 188 164 0.15 415 10 −8 19.5 13031.4 0.003 361 11 2.67 4.3 22.1 28.5 0.01 311 298 12 11 6.3 6.3 169 650717 306 254 254 13 11.9 14.8 100 122 >100 >100 367 366 14 0.89 2.80 2.952 at 0.16 <0.01 343 365 365 200 μM 15 2.05 6.8 163 133 <0.01 <0.03 315349 19 5.56 141 369 >100 24 298 20 7.66 26.0 26.0 181 149 0.01 330 330¹IU-NH-nuccloside hydrolase from Crithidia fasiculata: IAG-NH,IAG-nucleoside hydrolase from Trypanosoma brucei brocei. ²Where nohydrolysis was apparent, the slope was assumed to be less than 0.0001ΔOD/min. ³Changes in absorbance for the conversion 1 mM of theβ-D-ribofuranoside substrate to 1 mM product at the indicated pH anwavelength and for a 1 cm pathlength, calculated from experimentallyobserved data. The wavelength was the absorbance maximum. ⁴Absorbancedetermined experimentally for 1 mM solution of the phenolic aglycon atthe indicated pH and wavelength, and for a 1 cm pathlength.

Example 24 Detection of Giardia intestinalis

Giardia intestinalis (Portland 1 strain) was grown for 2 days in TYI-33medium supplemented with 5 mM arginine, harvested and washed. Asuspension containing 2.91×10⁸ cells of Giardia intestinalis in 1 mL ofaqueous HEPES buffer (40 mM. pH 7) was mixed with 1 mL of aqueous HEPESbuffer (40 mM, pH 7) containing 0.1 mM protease inhibitor E-64 andsonicated (Branson sonicator, duty cycle 40, output 2.5), for 2-3 min inice. The suspension was then centrifuged in a Beckman microfuge for 10mins at 4° C., and the supernatant decanted.

A second suspension of 2.91×10⁸ cells of Giardia intestinalis in 1 mL ofaqueous HEPES buffer (40 mM, pH 7) was mixed with 1 mL of 0.5% aqueousTriton X-100, left at 4° C. overnight, and the supernatant was decanted.

Aliquots (between 2 and 100 μL) of each of these supernatants were madeup to a volume of 1 mL of water containing 50 mM HEPES buffer at pH 8,0.24 mM 4-nitrophenyl β-D-ribofuranoside or 4-pyridylβ-D-ribofuranoside. The release of the aglycon was monitored byabsorption spectrometry at 400 nm or 253 nm, respectively. The 5 μLaliquots were sufficient for ready measurement of increased absorbance.The activity of the supernatant was stable to storage at 4° C. for 6days.

The rates of reaction were approximately the same for both substrates.For 4-nitrophenyl β-D-ribofuranoside, with an extinction coefficient for4-nitrophenol of 14,000, the activity was 470 nmoles.min⁻¹ per 10⁸cells, corresponding to an activity for the enzyme of 70nmoles.min⁻¹.(mg protein)⁻¹. For 4-pyridyl β-D-ribofuranoside, theactivity was 210 nmoles.min⁻¹ per 10⁸ cells, corresponding to anactivity for the enzyme of 31 nmoles.min⁻¹.(mg protein)⁻¹. Protein wasdetermined by Lowry method (ie Folin reaction).

Example 25 Detection of T. vaginalis

T. vaginalis was grown for 2 days in TYM medium, harvested, and washed.An extract of a suspension containing 7.76×10⁴ cells.mL⁻¹ T. vaginalisin aqueous HEPES buffer (40 mM, pH 7) was prepared using Triton X-100 asdescribed for Giardia intestinalis in Example 24.

The ability of the extract to hydrolyse substrates was determined as inExample 24. For 4-nitrophenyl β-D-ribofuranoside, the activity was 165nmoles.min⁻¹ per 10⁸ cells, corresponding to an activity for the enzymeof 9 nmoles.min⁻¹.(mg protein)⁻¹. For 4-pyridyl β-D-ribofuranoside, theactivity was 74 nmoles.min⁻¹ per 10⁻⁸ cells corresponding to an activityfor the enzyme of 4 nmoles.min⁻¹.(mg protein)⁻¹.

References

1. Lee H C; Galione, A; Walseth, T F Vitamins and Hormones 1994, 48,199.

2. Mentch, F; Parkin, D W; Schramm, V L Biochemistry 1987, 26, 921.

3. Horenstein, B A; Parkin, D W; Estupinan, B; Schramm, V L Biochemistry1991, 30, 10788.

4. Kline, P C; Schramm, V L Biochemistry 1995, 34, 1153.

5. Parkin D W; Horenstein, B A: Abdulah D R; Estupinan B; Schramm, V L JBiol Chem 1991, 266, 20658.

6. Estupinan, B; Schramm V L J Biol Chem 1994, 269, 23068.

7. Parkin, D W; Schramm V L Biochemistry 1995, 34, 13961.

8. Leung, H B; Kvalnes-Krick K L; Myer S L; deRiel, J K; Schramm V LBiochemistry 1989, 28, 8726.

9. Leung, H B; Schramm V L J Biol Chem 1980, 255, 10867.

10. Kline, P C: Schramm V L Biochemistry 1992 31, 5964.

11. Parkin, D W, J Biol Chem 1996, 271, 21713.

12. Kline, PC; Schramm V L Biochemistry 1993, 32, 13212.

Aspects of the invention have been described by way of example only andit should be appreciated that modifications and additions thereto may bemade without departing from the scope of the invention.

What is claimed is:
 1. A kit for detecting a nucleoside hydrolase or anucleoside phosphorylase in a sample comprising a chromogenic substratehaving the formula:

wherein X is OH or H, and Y is the residue of Y—OH, wherein Y—OH is achromophore or a compound readily converted to a chromophore.
 2. The kitof claim 1 which further comprises a cell-lysing agent.
 3. A dipstickfor detecting a nucleoside hydrolase or a nucleoside phosphorylase in asample comprising a chromogenic substrate having the formula:

wherein X is OH or H, and Y is the residue of Y—OH, wherein Y—OH is achromophore or a compound readily convened to a chromophore.
 4. Acompound having the formula:

wherein X is OH or H and Y is the residue of Y—OH, wherein Y—OH is achromophore or a compound readily convened to a chromophore, providedthat when X is OH, Y is not p-nitrophenyl, 1-naphthyl, 4-aminophenyl,5-amino-6-chloro-3-pyridazinyl, 4-chlorophenyl, phenyl, 4-acetylphenyl,4-methoxy phenyl, 4-hydroxyphenyl, 4-(N,N,N-trimethylammonio)phenyl; andprovided also that when X is H, Y is not 4-methylumbelliferyl; andprovided that the compound is not β-D-ribofuranoside of L-DOPA orL-α-methyl-DOPA or their N-acetyl methyl ester derivatives.
 5. Thecompound of claim 4 wherein Y is an optionally substituted pyridyl groupor a nitrophenyl.
 6. The compound of claim 4 which is selected from2-nitrophenyl β-D-ribofuranoside, 4-methylumbelliferylβ-D-ribofuranoside, 3-pyridyl β-D-ribofuranoside, 4-pyridylβ-D-ribofuranoside, 2-(5-nitropyridyl) β-D-ribofuranoside,β-D-ribofuranoside of luminol, 3-carboxamido-6-pyridylβ-D-ribofuranoside and 4-formylphenyl β-D-ribofuranoside.